Breast cancer is the most common malignancy in females in North America, becoming clinically apparent in one out of nine women. The prevalence of breast cancer is high compared to its annual incidence in other countries (estimated at greater than four times that in the UK). Thus, a therapy for this disease would provide a significant benefit to a large number of individual patients as well as a relief of health care resources.
Current treatment for breast cancer is directed at non-specific elimination-resection, administration of agents toxic to growing cells, or inhibition of receptor ligands required for cell growth. Examples include radiation, cytotoxic chemotherapy (e.g., doxorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, mitomycin C and mitoxantrone) or hormonal manipulation to delete (e.g., ovarian ablation) or antagonize (e.g., tamoxifen, aromatase inhibitors) estrogen/progesterone stimulation of tumor growth. Because of their lack of tumor specificity, these therapies are poorly tolerated and become ineffective when the disease is widely metastatic.
These therapies have additional limitations. High doses of cytotoxic agents needed for therapeutic efficacy also destroy normal dividing immunological cells and gastrointestinal cells. Thus, administration of cytotoxic agents is limited by neutropenia, thrombocytopenia and malnutrition. Radiation and surgical therapies are limited to relatively-localized disease. All strategies are limited by the degree of deformity and/or disability that patients are willing to tolerate for only a modest increase in survival. Thus, there is need for a therapy which specifically targets a patient's malignancy and does not reduce the quality of the patient's remaining life.
Breast cancers are difficult targets because they are heterogeneous in a variety of features, including, for example, presence and absence of estrogen and progesterone receptors, and presence and absence of amplified growth factor receptors. In addition, the tumor cells may have a variety of different mutations in somatic proto-oncogenes, such as c-erb2, c-myc, int2, hst1, bcl1, and PRAD1, or tumor suppresser genes, including RB and TP53. Further, a patient may have more then one malignant cell type in the same tumor.
A population of diversified targets however, is exactly what a host's immune system is designed to screen and selectively eliminate. Vaccine-based immunotherapy has been shown to be effective in treating animal models of other types of cancers. The diversity of targets in breast cancer plus effectiveness in other types of cancer suggests that development of a vaccine-based immunotherapy might be effective for the treatment of breast cancer.
Antisense Oligonucleotides
Antisense oligonucleotides are nucleotide sequences that are complementary to specified segments of a targeted gene or mRNA. The binding of an antisense oligonucleotide to DNA or RNA within a cell can inhibit translation or transcription in the cell, which can disrupt gene expression. Typically, antisense oligonucleotides are about 14 to about 25 nucleotides in length, since it is believed that at least about 14 bases are required to specifically target a unique mammalian gene sequence. The sequence of an antisense oligonucleotide is chosen to provide specificity for a particular mammalian gene or mRNA sequence.
Antisense oligonucleotides can be synthesized with either natural or synthetic bases and with a natural phosphate or modified phosphate or sugar backbone. For example, phosphothioate, phosphonate, and other backbone modifications can significantly alter the biological half-life, and bioavailability of antisense oligonucleotides.
Advances in molecular biology and synthetic chemistry over the past two decades have stimulated interest in developing antisense oligonucleotides as therapeutic agents. It would be beneficial to develop an antisense oligonucleotide based therapy for breast cancer to provide targeted immunotherapy.
Insulin-Like Growth Factor and its Receptor
Growth factors and their receptors are examples of molecular switches whose activation transduces a signal to the cell nucleus that enables growth, transformation and protection from cell death. Insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) appear to be required for mitosis in many cell types. In vitro, most cells in culture are dependent on IGF-1 for growth, and many tumor lines secrete IGF-1 and express IGF-1R. IGF-1R is required for the entry of stimulated lymphocytes and HL-60 cells into S Phase. In the absence of proliferation, such as in senescent human fibroblasts, IGF-1 mRNA is not detectable by reverse transcriptase polymerase chain reaction. Once these senescent cells are transfected with a temperature sensitive SV40 T antigen gene, they regain the ability to express IGF-1 mRNA at permissive temperatures. IGF-1R is also associated with growth in vivo. Mice with homozygous null mutations for igf-1r gene die shortly after birth with a body weight 30% of wild type.
The expression of IGF-1R may be required for transformation in vitro and for tumor maintenance in vivo. When the gene encoding IGF-1R is disrupted in mouse embryo fibroblasts, transformation by either Ha-ras, SV40 tumor antigen or both, is prevented. The transformed phenotype is restored once cells are transfected with a plasmid expressing the IGF-1R RNA. Overexpression of the IGF-1R results in increased transformability of NIH 3T3 cells. Not only is oncogenesis associated with the induction of IGF genes, but preliminary data suggests that the gene product of the retinoblastoma tumor suppresser gene inhibits the expression of the IGF-1 gene.
The IGF-1 receptor (IGF-1R) is a membrane glycoprotein composed of two alpha (Mr 130,000) and two beta (Mr 98,000) subunits linked together by disulfide bonds. The alpha subunit binds IGF-1 and IGF-2 with equal affinity. The .beta.-subunit has an intracellular domain with tyrosine kinase activity which upon activation by either IGF-1 or IGF-2 autophosphorylates its own .beta.-subunit and two major substrates, insulin receptor substrate 1 and Shc. Once activated, IGF-1R transmits a signal, transduced through ras and raf, to the nucleus. IGF-1 is known to stimulate the expression of approximately 30 genes expressed in 3T3 cells, encoding both cytoplasmic and nuclear proteins. Within one minute of IGF-1R ligand binding, a series of other cellular proteins as well as nuclear proteins including the 43 kD product of the c-jun protooncogene, are also phosphorylated in vitro.
The IGF-1R is required for the action of several growth factors. Antisense to IGF-1R blocks the EGF stimulated proliferation of 3T3 cells overexpressing EGFR. Neither PDGFR nor EGFR antisense inhibit IGF-1 stimulated growth in cells overexpressing IGF-1R. There is evidence that other protooncogenes, such as c-myb, induce expression of IGF-1 and IGF-1R. When c-myb is overexpressed in fibroblasts, the IGF-1 requirement for growth is lost because both IGF-1 and IGF-1R mRNA is induced. When c-myb expression is inhibited, there is a decrease in the IGF-1R mRNA. However, inhibition of IGF-1R expression has no effect on the c-myb mRNA levels.
IGF-1R antisense can inhibit the growth of cells whose growth depends on expression of IGF-1R. Cells that are exposed to IGF-1R antisense and cells that are transfected with a viral vector expressing IGF-1R antisense show diminished levels of IGF-1R protein. This results in growth inhibition in IGF-1R dependent cells like rat C6 glioblastoma cells, an IGF-1R dependent tumor cell line. Diminished tumorogenicity results from transfection of C6 glioblastoma cells with a viral vector expressing IGF-1R antisense. For example, no tumors developed in rats injected with C6 glioblastoma cells bearing a viral vector expressing IGF-1R antisense. Injection with these antisense-transfected cells can also protect rats from glioblastoma tumor formation due to subsequent injection of wild type C6 glioblastoma cells. No tumors appeared in 4 weeks after the injection of the wild-type cells. Injection of the antisense-transfected C6 glioblastoma cells also caused regression of previously established wild-type C6 glioblastoma tumors. In each of these experiments the cells that exhibited diminished tumorogenicity or that affected tumor growth were transfected with a viral vector to express IGF-1R antisense.
Work, to date, studying IGF-1R antisense in cancer cells has been performed by transfecting cells with antisense DNA integrated in a viral vector. The consequences of inserting viral DNA into human gliomas are unknown. An estimated 23 human years of retroviral mediated gene transfer has been performed in humans without known side effects, although some of the viral vectors being used for gene therapy have the long-term potential for causing cancer. Side effects have been described in 3 monkeys at the NIH, who developed malignant T cell lymphoma after bone marrow transplant and gene transfer with a helper virus contaminated retrovirus. Aside from the potential side effects and risks of viral contamination, using viral vectors to transfect each tumor line with antisense is very labor intensive.
The practical use of antisense genetic therapy would be greatly enhanced by incorporation of antisense oligonucleotides into the breast, or other, tumor cells without the use of a retroviral vector. In light of the present shortcomings in breast cancer therapy, it would be advantageous to have a therapy that specifically inhibited the growth and metastasis of breast cancer cells without the side effects of present therapies. Antisense oligonucleotides present one avenue for such therapy, but present methods for treatment with antisense oligonucleotides require viral vectors and have not proven effective in inhibiting the growth and metastasis of breast cancer cells. Hence, there is a need for an antisense-oligonucleotide based therapy for breast cancer that eliminates use of viral vectors.